The staining of biological tissues and cells with dyes, especially fluorescent dyes, in order to differentiate one type from another or to render them more observable, is well known in the art. Dyes that fluoresce in the red region of the electromagnetic spectrum are especially desirable.
Further, chemical analysis of liquids, such as water, milk and biological fluids is often desirable or necessary for health maintenance and diagnostic care. Various compositions and elements to facilitate such analyses are known. Such compositions and elements generally include a reagent composition for determining a substance under analysis, identified as an "analyte" herein. The analyte can be a living organism, such as a microorganism or yeast cell, or a nonliving chemical substance. The reagent composition, upon interaction with the analyte, provides a detectable change (e.g. dye formation).
Recently, much work has been directed to developing compositions and elements which are useful for rapid and highly quantitative diagnostic or clinical analysis of biological fluids such as whole blood, blood sera, plasma, urine and the like.
For example, for the rapid and effective diagnosis and treatment of infectious diseases, it is desirable to be able to detect the bacteria causing the disease as rapidly as possible. Infections of the urinary tract are among the most common bacterial diseases, second in frequency only to infections of the respiratory tract. Most urinary tract infections are associated with bacterial counts of 100,000 or more organisms per ml of urine, a condition referred to as significant bacteriuria
In the Belly et al patent application noted above, i.e. U.S. Ser. No. 824,766, novel reducible compounds are described and claimed which are useful in the detection of bacteria as well as other oxidizable analytes. The assays described in that application can be used to determine an analyte, e.g. a bacterium, which reduces a reducible compound thereby releasing a detectable species, e.g. a colored or fluorescent dye, at a pH of 9 or less.
While the assays of Belly et al represent a significant advance in the art, it is desirable to improve the sensitivity of the assays even further to extend their usefulness with analytes at very low concentrations. It is generally known that assays using fluorescent dyes are more sensitive than those using colorimetric dyes. A variety of fluorescent assays have been developed, most of which use coumarin or unbelliferone derivatives as the fluorescent dyes. In the Belly et al application, 4-methylumbelliferone, a representative umbelliferone derivative, was attached to a quinone nucleus to form a reducible compound according to that invention. However, our studies have shown that the resulting compound had limited stability to light (see Example 13 below).
Other known fluorogens present other problems. Some fluorescent dyes can be used only at high pH (greater than 9). They can not be used in biological assays which are normally carried out at lower pH. Further, coumarin or umbelliferone dyes, emit fluorescence at wagvelengths at which spectral interferents can be significant, i.e. at wavelengths below 500 nm. This characteristic further limits their usefulness in biological assays.
Recently, improved fluorescent unbelliferone derivatives have been described (Wolfbeis et al, Bull. Chem. Soc. Japan, 58:731, 1985) and used in an acid phosphatase assay (Anal. Biochem., 143:146, 1984). Some of these dyes are reported to have pKa values of about 6, and fluorescence emissions at 595 nm (at pH 9). However, these dyes exhibit absorption below about 500 nm (one dye absorbs at 505 at pH 9), i.e. in the same region that some serum components, such as hemoglobin and bilirubin, have strong spectral absorptions.
Hence, there is a need for a highly sensitive assay which is not subject to the problems noted above.